Analogs of human basic fibroblast growth factor mutated at one or more of the positions glutamute 89, aspartate 101 or leucine 137

ABSTRACT

The present invention relates to muteins of human basic fibroblast growth factor with superagonist properties. Both protein and the respective encoding nucleic acid species are disclosed. The invention also embodies vectors and host cells for the propagation of the nucleic acid sequences and the production of the muteins.

This application claims the benefit of the filing date of provisionalapplication No. 60/068,667 filed on Dec. 23, 1997, which is hereinincorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the identification of new muteins ofhuman basic fibroblast growth factor that are unusually potentstimulators of cell division.

2. Description of the Related Art

Fibroblast growth factors (FGFs) are an evolutionarily conserved, largefamily of mitogenic proteins that stimulate mitosis in mesodermal andneuroectodermal cell lineages (Basilico, C. and Moscatelli, D., Advancesin Cancer Research 59:115-165, Eds. Vande Woude, G. F. and Klein, G.(1992)). These proteins also bind heparin and are often referred to inthe literature as heparin binding growth factors (HBGFs). Family membersshare a high degree of nucleic acid and amino acid sequence homology.

Complementary DNA clones encoding basic FGF (bFGF), one member of theFGF family, have been isolated and sequenced. The protein is found tohave 89 to 95% amino acid identity among several species, includinghuman, bovine, and rat (Xenopus bFGF is more divergent, sharing 80%homology with human bFGF). This degree of conservation suggests that allregions of the protein may be functionally important. Inhumans, bFGF isexpressed in four forms: (1) an 18-KDa form (155 amino acids) initiatedfrom an AUG codon; (2), (3) and (4) that are 22, 22.5 and 24 kDA,respectively, and all initiated from the CUG codons resulting inN-terminal extensions of varying lengths relative to the 18 kDa form(Florkiewicz, R. Z. and Sommer, A., Proc. Natl. Acad. Sci. (USA)86:3978-3981 (1989); Pratts, H., et al., Proc. Natl. Acad. Sci. (USA)86:1836-1840 (1988)). Additionally, while the different forms of bFGFare localized in different compartments of the cell, there is onlylimited information relating to the functional significance of suchsubcellular localization. The biological activity of bFGF on cells intrans is effected through signal transduction after cell surface bindingto an FGF specific receptor and to heparin sulfate proteoglycans(Moscatelli, D., J. Cell Physiology. 131:123-130 (1987)). In all tissuesso far examined, bFGF is found to be expressed, perhaps reflecting itsbroad spectrum mitogenic activity.

Due to its ability to stimulate the proliferation of a wide variety ofcell types, bFGF plays a significant role in many biological processes:(1) angiogenesis (Folkman, J. and Klagsbrum, M., Science 235:442-447(1987); (2) wound healing (Slavin, J., J Pathology 178: 5-10, 1996;McGee, G. S., et al., J. Surg. Research n45:145-153 (1988); (3)embryogenesis (Kimelman, D., et al., Science 242:1050-1056 (1988);Herbert, J. M., et al., Dev. Biol. 138:454-463 (1990); and (4)tumorigenesis (Ulrich, R., et al., Cancer Cell 3(8) :308-311 (1991);Nguyen, M., et al., J. Natl. Cancer. Inst. 86(5):356-361 (1994); Wright,J. A., et al., Crit. Rev. Oncogenesis 4(5):473-492 (1993)). Also thereis evidence indicating that bFGF may be used therapeutically in thetreatment of cerebral ischemia (Lyons, M. K., et al., Brain Research558:315-320 (1991); Koketsu, N., et al., Annals Neurology35(4):451-457(1994), cerebral aneurysyms (Futami, K., et al., Stroke26(9):1649-1654 (1995) and neural injury (Logan, A. and Berry, M., TIPS14:337-343 (1993); Eckenstein, F. P., J. Neurobiology 25(11):1467-1480(1994); Gomez-Pinilla, F., et al., J. Neuroscience 15(3):2021-2029(1995)), in addition to its therapeutic potential in the treatment ofvascular disease (Richard, J-L., et al., Diabetes Care 18(1):64-69(1995); Lindner, V., et al., J. Clin. Invest. 85:2004-2008 (1990);Lazarous, D. F., et al., Circulation 91(1):145-153 (1995)) and gastricand duodenal ulcers (Folkman, J., et al., Ann. Surg. 214(4):414-427(1991); Szabo, S., et al., Scand. J. Gastroenterology 30 Suppl. 208:3-8(1995); Kitijima, M., et al., Microvasc. Research 50:133-138 (1995);Kusstatscher, S., et al., J. Pharm. Exp. Therapeutics 275:456-461(1995); Sazbo, S., et al., Gastroenterology 106:1106-1111 (1994);Konturek, S. J., et al., Gut 34:881-887 (1993)).

In order to more fully understand this widespread, biologicallysignificant ligand-receptor system, basic research in this field isfocused on elucidating the relationship between bFGF protein structureand function. Early structural studies utilized synthetic peptidescorresponding to different regions of the bFGF protein to grossly mapthe heparin binding and receptor binding regions of the protein (Bairdet al., Proc. Natl. Acad. Sci. (USA) 85:2324-2328 (1988); Baird et al.,J Cell Phys. Suppl. 5:101-106 (1987)). More recently, high resolutionX-ray crystallography studies (Zhu et al., Science 252:90-93 (1991);Zhang et al, Proc. Natl. Acad. Sci. (USA) 88:3446-3450 (1991); Erikssonet al., Proc. Natl. Acad. Sci. (USA) 88:3441-3445 (1991)) have been usedin structure-based, site-directed mutagenesis analyses (Thompson et al.,Biochemistry 33(13):3831-3840 (1994); Springer et al., J. Bio. Chem.269(43):26879-2688 (1994)) and biophysical characterizations of theinteractions of bFGF with the bFGF receptor and heparin (Pantoliano etal., Biochemistry 33:10229-10248 (1994)) to further define thefunctional domains of the protein.

The studies of Thompson et al., Springer et al. and Pantoliano et al.have established the following: (1) the primary receptor binding domain(site 1) is a discontinuous domain, important points of contact beingamino acids Y24, R44, N101, Y103, L140 and M142, which are exposed tosolvent; (2) a secondary receptor binding domain (site 2) (approximately250 fold weaker binding) is important for bFGF mitogenicity andcomprises amino acids 106-115, which form a type-1 β-turn; and (3) theheparin binding domain which is also a discontinuous domain, the keyamino acids of which are K26, N27, R81, K119, R120, T121, Q123, K125,K129, Q134 and K135. (Note: letter/number designations correspond to thesingle letter amino acid code followed by the position in the linearamino acid sequence for bFGF as described by Zhang et al., 1991.)

One current model of bFGF action suggests that the monomeric ligand bFGFbinds to its cell surface receptor through both the high affinity domain(site 1) and the low affinity domain (site 2), leading to receptordimerization and signal transduction. Heparin binding, known to beimportant for bFGF activity, is believed to promote site 2 binding tothe receptor. (Pantoliano et al., Biochemistry 33:10229-10248 (1994)).

The amino acid sequence of wild-type bFGF is disclosed in severalpublications: for example, U.S. Pat. Nos. 5,155,214; 4,994,559;5,439818; 5,604,293; European Patent Publication No. EP 0 237 966 A2, toname a few.

Structure/function information is useful in studying the biologicalactivity of variants of the bFGF protein sequence. Consequently, thereis a great deal of interest in generating new muteins of bFGF for study.For example, analogs in which at least one amino acid is substituted,preferably targeting Cys, Asp, Arg, Gly and Val residues, have beenreported (International Publication No. WO 91/09126). Anotherpublication describes a replacement mutein in which at least onecysteine residue is substituted with another amino acid, and deletionmutations in which either 41 amino acids are missing from the aminoterminus or 61 amino acids are missing from the carboxyl terminus (U.S.Pat. No. 5,478,740). Mutations in the heparin binding domain of humanbFGF are known to alter its biological activity (Heath et al.,Biochemistry 30(22):5608-5615 (1991)) and the highly conserved Arg 40and Arg 45 residues are necessary for stability and mitogenicity of bFGF(Arakawa et al., J. Protein Chemistry 14(5):263-274 (1995)).Additionally, enhanced stability analogs have been reported in which 2or 3 amino acids are added, deleted or substituted, with serinesubstitution being preferred for cysteine residues (Eur. Pat. Pub. No.EP 0 281, 822 B2).

As previously disclosed, bFGF is a powerful mitogen and a key regulatoryfactor in many biological processes: for example angiogenesis, woundhealing, ischemic tissue repair, gastric and duodenal ulcer healing,tumorigenesis and neural tissue survival and regeneration. Notsurprisingly, the therapeutic value of wild-type and mutein bFGF's isrecognized and detailed in the art, some examples of which are listedherein. Therapeutic treatments related to the above disclosed biologicalprocesses have been described for wild-type bFGF in U.S. Pat. Nos.5,612,211; 5,439,818; 5,604,293; 5,514,566; 4,994,559; 5,514,662 andEuropean Patent Application No. EP 0 237 966 A2. Similar therapeutictreatments utilizing bFGF muteins are disclosed in U.S. Pat. Nos.5,132,408; 5,352,589; 5,360,896; 5,371,206; 5,302,702; 5,310,883;5,478,804; 5,576,288 and European Patent Application No. EP 0 281 822A2. For example, replacement muteins in which the loop region of humanbFGF (amino acid residues 118-122) are replaced with selected peptidesof other FGF family members are described in U.S. Pat. No. 5,491,220.These muteins are disclosed to be useful in the treatment of cancer asantiproliferative agents or as agents that promote vascularization.

Given the potential therapeutic value of the bFGF protein, there is aneed in the art for the development of novel analogs of bFGF withimproved biological properties.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide analogs ofhuman bFGF with superagonist activity. Other objects, features andadvantages of the present invention will be set forth in the detaileddescription of the preferred embodiments that follows, and in part, willbe apparent from the description or may be learned by practice of theinvention.

In a first embodiment, the present invention is directed to muteins ofhuman bFGF in which Glutamate 89 or Aspartate 101 or Leucine 137 orcombinations or permutations thereof are substituted with a neutraland/or hydrophobic amino acid. Other embodiments are drawn topolynucleotides encoding the muteins of the first embodiment, a vectorcontaining said polynucleotide and a host cell carrying said vector. Athird group of embodiments are drawn to processes to produce apolypeptide, to produce cells capable of producing said peptide and toproduce a vector containing DNA or RNA encoding said polypeptide. Afourth group of embodiments are drawn to methods to stimulate celldivision, to heal a wound, to treat ischemia, to treat peripheralvascular disease, to treat a gastric or duodenal ulcer, to treat neuralinjury and a pharmacologic composition comprising an effective amount ofthe mutein of the first embodiment.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. This figure presents the DNA (SEQ ID NO:1) and amino acid (SEQID NO:2) sequence of the 157 amino acid form of bFGF used in thisinvention. The N-terminal, initiating methionine is processed by E.coli, and the purified bFGFs, therefore, lack the first amino acid shownin the figure. The nucleic acid sequence reported in this figure isrepresentative of the wild-type bFGF DNA sequence followingmodifications of the gene purchased from R&D Systems. The DNAmodifications were performed to incorporate restriction sites forsubdloning and cassette mutagenesis; in all cases, the original aminoacid sequence remained unaltered. The boxed and numbered amino acidsidentify those subjected to site-directed mutagenesis.

For reference purposes, the glycine at position 1 of this sequencecorresponds to the first amino acid in the sequence reported by Sommeret al., Biochem. Biophys. Res. Commun. 144(2):543-550 (1987)), themethionine at amino acid position 3 of this sequence corresponds to thefirst amino acid reported for the 18 kDA form of human bFGF reported byPrats et al, Proc. Natl. Acad. Sci. (USA) 86(6):1836-1840 (1989);GenBank Accession No. JO4513) and the proline at position 12 of thissequence corresponds to the first amino acid in the human bFGF sequencereported in FIG. 4 of U.S. Pat. No. 5,439,818 by Fiddes et al.

FIG. 2. This figure displays the results of a single mitogenicityexperiment in which the wild-type and mutant bFGFs were all assayedsimultaneously on Swiss 3T3 fibroblasts and is representative of thedata compiled in Table I.

The concentration of wild-type and mutant bFGF required to give the ½maximal response (ED₅₀) was plotted and calculated using the followingequation: y=(m*x/(x+c)), where y=absorbance, x=concentration of growthfactor, m=the maximum response and c=ED₅₀. During each assay on mutantproteins, a parallel analysis was performed for wild-type bFGF. Ourrecombinant human wild-type bFGF expressed and purified from E. coli hasbeen repeatedly shown to have an ED₅₀ value identical to the bFGFinternational standard (code 90/712) obtained from the NationalInstitute for Biological Standards and Control.

Numbering of amino acids is based on the expression of a 157 amino acidform of bFGF as described in the Examples section and in FIG. 1. Aminoacids are identified using the single letter code. Mutations aredesignated by the one letter code for the original amino acid, followedby the amino acid number, followed by the one letter code for thereplacement amino acid.

The dose response data presented in FIG. 2 demonstrates that the ED₅₀values for the bFGF muteins are significantly lower than that ofwild-type bFGF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first embodiment, the present invention is drawn to a mutein ofhuman basic fibroblast growth factor (bFGF), or biologically activepeptide thereof, in which a neutral and/or hydrophobic amino acid hasbeen substituted for one or more of the following amino acids: Glutamate89, Aspartate 101 or Leucine 137.

A human bFGF mutein is defined as comprising human basic fibroblastgrowth factor in which at least one amino acid of the native protein hasbeen substituted by another. Generally speaking, a mutein possesses somemodified property, structural or functional, of the native protein. Forexample, the mutein may be antagonistic or agonistic with respect tonormal properties of the native protein. An antagonist is defined as asubstance that to some extent nullifies the action of another. Forexample, a molecule that binds a cell receptor without eliciting abiological response. An agonist is defined as a substance that induces acellular or physiologic response. For example, a molecule that binds toa receptor and elicits a biological response. A biological response maybe, for example, the stimulation of cell division, chemotaxis,angiogenesis or wound repair. A biological response may encompass otherfunctional properties of the native protein and would be well known tothose practicing the art. As used herein, these terms are not limiting,it being entirely possible that a given mutein is antagonistic to onebiological response and agonistic with respect to another.

More specifically, the first preferred embodiment of the invention ishuman bFGF protein in which substitution means that any neutral and/orhydrophobic amino acid replaces at least one of the following: Glutamate89, Aspartate 101 or Leucine 137. For clarity, the human bFGF proteinsequence is presented in FIG. 1 to indicate exactly which amino acidresidues are mutated. A neutral amino acid is defined to include serine,threonine, alanine, asparagine, glutamine, cysteine, glycine andnon-naturally occurring analogues thereof. A hydrophobic arnino acid isdefined to include tyrosine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, methionine and non-naturally occurringanalogues thereof.

Because one, two or three bFGF residues are possibly mutated in the bFGFmutein of the invention, the following numerical permutations arepossible for mutein protein structure, where X stands for any neutral orhydrophobic amino acid, or non-naturally occurring analogue thereof:human bFGF [X⁸⁹], human bFGF [X¹⁰¹], human bFGF [X¹³⁷], human bFGF[X⁸⁹,X¹⁰¹], human bFGF [X⁸⁹, X¹³⁷], human bFGF [X¹⁰¹,X¹³⁷] and humanbFGF [X⁸⁹,X¹⁰¹,X¹³⁷].

Therefore, in the first embodiment of the invention, X is defined as anyneutral or hydrophobic amino acid. Consequently, where a mutein containsmore than one of the described amino acid residue mutations, thesubstituted amino acid at a given particular mutated site may be aneutral or hydrophobic amino acid. For example, in one claimedembodiment of the invention, a neutral amino acid is defined as alanine,and a hydrophobic amino acid is defined as tyrosine. Thus, it will beobvious to one skilled in the art, referring to the above describednumerical permutations for the bFGF mutein structure, that the number ofpossible amino acid sequence permutations depends on how X is defined:for example, human bFGF [Ala⁸⁹,Tyr¹⁰¹], [Tyr⁸⁹,Ala¹⁰¹] and others areindicated when X, a neutral or hydrophobic amino acid, is defined asalanine or tyrosine. In a more specific form of the first embodiment,the mutein of human basic FGF comprises the substitution of alanine forat least one of the following: Glutamate 89, Aspartate 101 or Leucine137. The following muteins are indicated in the specific embodiment foralanine substitution identified above: human bFGF [Ala⁸⁹], human bFGF[Ala¹⁰¹], human bFGF [Ala¹³⁷], human bFGF [Ala^(89,101) ],human bFGF[Ala^(89,137)], human bFGF [Ala^(101,137)] and human bFGF[Ala^(89,101,137)].

In another more specific form of the first embodiment, the mutein ofhuman basic FGF comprises the substitution of tyrosine for at least oneof the following: Glutamate 89, Aspartate 101 or Leucine 137. Thefollowing muteins are indicated in the specific embodiment for tyrosinesubstitution identified above: human bFGF [Tyr⁸⁹], human bFGF [Tyr¹⁰¹],human bFGF [Tyr¹³⁷], human bFGF [Tyr^(89,101)], human bFGF[Tyr^(89,137)], human bFGF [Tyr^(101,137)] and human bFGF[Tyr^(89,101,137)].

The mutein may be produced by any method known to those skilled in theart. These methods would include, but are not limited to, laboratorymeans such as encompassed by recombinant DNA technologies or through thechemical synthesis of human bFGF possessing a neutral or hydrophobicamino acid at one or more of the designated positions (Glutamate 89,Aspartate 101 or Leucine 137).

The mutein of the present invention is also seen to include any and allmutations known to those in the art so long as at least one of theresidues identified in the invention is appropriately mutated. Examplesof mutations in the art include the following: deletion mutein bFGF1-146, bFGF [Ala⁶⁹, Ser⁸⁷], bFGF [Arg¹⁹], bFGF [Glu¹⁰⁷], bFGF [Glu¹²³],bFGF [Ile¹⁰⁷], bFGF [Glu^(107,123)] and bFGF [Arg¹⁹, Lys^(123,137)] inHeath, W. F., et al., Biochemistry 30:5608-5615 (1991)); bFGF[Ser^(69,87)] in PCT Pub. WO 91/09126; bFGF [Gln¹²⁸], bFGF [Gln¹³⁸],bFGF [Gln^(128,138)], bFGF [Gln¹²⁹], bFGF [Gln¹³⁴], bFGF [Gln^(129,134)]in Lu-Yuan, L., et al., Biochemistry 33:10999-11007 (1994)); bFGF loopreplacement muteins (residues 118-1 22) in Seddon, A. P., et al.Biochemistry 34:731-736 (1995)); bFGF [Ala⁹⁶], bFGF [Ala¹⁰⁷], bFGF [Alaor Phe at 109-114] in Zhu, H., et al., J. Biol. Chem.270(37):21869-21874 (1995)). These mutations possess a certain mitogenicpotential or receptor or heparin-binding activity that may or may notprovide a basis for antagonistic or agonistic activity, which incombination with the mutations of this invention, will provide for abFGF molecule with unique properties. Additional muteins with specificmutations are known and others will be developed. The mutein of thisinvention may incorporate one or more of the mutations that are commonlyknown in the art at the time.

The most preferred method for producing the mutein is throughrecombinant DNA methodologies and is well known to those skilled in theart. Such methods are conveniently found in published laboratory methodsmanuals such as Current Protocols in Molecular Biology (John Wiley &Sons, Inc.), which is incorporated herein by reference. For example, anypreviously isolated clone encoding human basic FGF, or variants thereof,will serve as a starting point to introduce one or more of thedesignated substitutions utilizing the method of site-directedmutagenesis. The term “variants of bFGF” refers to all muteins disclosedin the prior art, as discussed above.

Additionally, the first preferred embodiment includes a biologicallyactive peptide derived from the mutein described herein. Such a peptidewill contain at least one of the substitutions described for the muteinand will possess biological activity. The peptide may be produced by anyand all means known to those skilled in the art, examples of whichinclude but are not limited to enzymatic digestion, chemical synthesisor recombinant DNA methodologies.

It is well established in the art that fragments or peptides of the bFGFprotein are biologically active. For example, see the heparin-bindingand receptor-binding experiments done by Baird et al., Proc. Natl. Acad.Sci. (USA) 85:2324-2328 (1988), and J. Cell. Phys. Suppl. 5:101-106(1987)). Furthermore, there is sufficient information relating proteinstructure to biological function for the bFGF protein that one ofordinary skill in the art knows how to select such peptides. Forexample, see bFGF schematic relating protein structure and function inBasilico, C. and Moscatelli, D., Advances in Cancer Research 59:115-165,Eds. Vande Woude, G. F. and Klein, G. (1992)).

Therefore, the selection of fragments or peptides of the mutein is basedon criteria known in the art. The first criterion is based on thestructural requirements for at least one of the mutations identified inthe invention and a sufficient amount of additional residues for areasonable expectation of biological function using structure/functioninformation in the literature. The second criterion is based on afunctional test to evaluate biological activity similar to what isalready known in the art, e.g. a mitogenic assay or heparin-bindingassay, such as the cell proliferation assay disclosed in Springer, B. A.et al., J. Bio. Chem. 269(43):26879-26884 (1994)) and the heparinaffinity chromatography disclosed in Thompson, L. D., et al.,Biochemistry 33(13):3831-3840 (1993)).

The present invention also encompasses a polynucleotide encoding theabove described mutein that may be in the form of RNA or in the form ofDNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNAmay be double-stranded or single-stranded. The coding sequence thatencodes the mutein of the present invention may vary as a result of theredundancy or degeneracy of the genetic code.

The polynucleotide that encodes for the mutein indicated in FIG. 1 mayinclude the following: only the coding sequence for the mutein; thecoding sequence for the mutein and additional coding sequence such as afunctional polypeptide, or a leader or secretory sequence or aproprotein sequence; the coding sequence for the mutein (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the mutein.Thus, the term “polynucleotide encoding a mutein” encompasses apolynucleotide which includes only coding sequence for the mutein aswell as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the describedpolynucleotide that encode for fragments, analogs and derivatives of thepolypeptide that contain the indicated substitutions. The variant of thepolynucleotide may be a naturally occurring allelic variant of the humanbFGF sequence or a non-naturally occurring variant. Thus, the presentinvention also includes polynucleotides encoding the mutein describedabove as well as variants of such polynucleotides which variants encodefor a fragment, derivative or analog of the disclosed mutein. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants as long as at least one of the indicatedamino acid substitutions is present. These nucleotide variants are mostreadily identified in the prior art as encoding muteins of the bFGFprotein (discussed above).

As indicated, the polynucleotide may have a coding sequence which is anaturally occurring allelic variant of the sequence shown in FIG. 1. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides wherein the codingsequence for the mutein may be fused in the same reading frame to apolynucleotide sequence that aids in expression and secretion of apolypeptide from a host cell. For example, a leader sequence thatfunctions as a secretory sequence for controlling transport of apolypeptide from the host cell. The polypeptide having a leader sequenceis a preprotein and may have the leader sequence cleaved by the hostcell to form the mature form of the polypeptide. The polynucleotides mayalso encode for a proprotein which is the mature protein plus additionalN-terminal amino acid residues. A mature protein having a prosequence isa proprotein and is an inactive form of the protein. Once theprosequence is cleaved an active mature protein remains. Thus, forexample, the polynucleotide of the present invention may encode themutein, or the mutein having a prosequence or the mutein having both aprosequence and a presequence (leader sequence).

The polynucleotide of the mutein also may encode for a chimeric moleculethat contains, in part or in whole, the mutein coding sequence and thecoding sequence of any other gene, or part thereof, known in the art.The nonmutein coding sequence may be positioned either 3′ or 5′ of themutein coding sequence. The resultant chimeric protein containsproperties of both the mutein and nonmutein protein. Examples of such achimeric protein include, but are not limited to, the coding sequence ofanother growth factor, a protein that stabilizes the FGF mutein or aprotein that targets the peptide to a particular tissue or cell type.

The mutein of the present invention may also have the coding sequencefused in frame to a marker sequence which allows for purification of themutein of the present invention. The marker sequence may be ahexa-histidine tag or the T7 peptide (amino acid sequence: Met Ala SerMet Thr Gly Gly Gln Gln Met Gly (SEQ ID NO:4)) supplied by a vector toprovide for purification of the polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell 37:767 (1984)). Othermarker sequences well known to those skilled in the art may be used forsimilar purposes.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site by procedures known in theart.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli lac or trp, the phagelambda P_(L) promoter, the T7 phage promoter and other promoters knownto control expression of genes in prokaryotic or eukaryotic cells ortheir viruses. The expression vector also contains a ribosome bindingsite for translation initiation and a transcription terminator. Thevector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence, as well as anappropriate promoter or control sequence, is employed to transform anappropriate host to permit the host to express the protein. Asrepresentative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Salmonella tyiphimurium, Streptomyces;fungal cells, such as yeast; insect cells, such as Drosophila and Sf9;animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. The following vectorsare provided by way of example: Bacterial-pET (Novagen), pQE70, pQE60,pQE-9 (Qiagen), pBs, phagescript, psiX174, pBlueScript SK, pBsKS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK2233, pKK233-3, pDR540,pRIT5 (Pharmacia); and Eukaryotic-pWLneo, pSV2cat, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Thus, these and anyother plasmids or vectors may be used as long as they are replicable andviable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include laci, lacz, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation, techniques well known in the art.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

The bFGF mutein can be expressed in mammalian cells, insect, yeast,bacterial, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., 1989, the disclosure of which is hereby incorporated byreference.

Transcription of a DNA encoding the mutein of the present invention byhigher eukaryotes is increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about from10 to 300 bp, that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100 to 270), a cytomegalovirus early promoter enhancer, apolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and the S.cerevisiae TRP1 gene, and a promoter derived from a highly-expressedgene to direct transcription of a downstream structural sequence. Suchpromoters can be derived from operons encoding glycolytic enzymes suchas 3-phosphoglycerate kinase (PGK), α factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion oftranslated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed for the expression of foreignproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

Various mammalian cell culture systems can also be employed to expressthe mutein. Examples of mammalian expression systems include the COS-7lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175(1981), and other cell lines capable of expressing a compatible vector,for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalianexpression vectors will comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements.

RNA vectors may also be utilized for the expression of the muteinsdisclosed in this invention. These vectors are based on positive ornegative strand RNA viruses that naturally replicate in a wide varietyof eukaryotic cells (Bredenbeek, P. J. and Rice, C. M., Virology3:297-310 (1992)). Unlike retroviruses, these viruses lack anintermediate DNA life-cycle phase, existing entirely in RNA form. Forexample, alpha viruses are used as expression vectors for foreignproteins because they can be utilized in a broad range of host cells andprovide a high level of expression; examples of viruses of this typeinclude the Sindbis virus and Semliki Forest virus (Schlesinger, S.,TIBTECH 11:18-22 (1993); Frolov, I., et al., Proc. Natl. Acad. Sci.(USA) 93:11371-11377 (1996)). As exemplified by Invitrogen's Sinbisexpression system, the investigator may conveniently maintain therecombinant molecule in DNA form (pSinrep5 plasmid) in the laboratory,but propagation in RNA form is feasible as well. In the host cell usedfor expression, the vector containing the gene of interest existscompletely in RNA form and may be continuously propagated in that stateif desired.

The mutein is recovered and purified from recombinant cell cultures bymethods established in the art, which include but are not limited toammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxyapatite chromatography and lectin chromatography. Proteinrefolding steps can be used, as necessary, in completing configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps.

Another embodiment of the invention relates to a method of stimulatingcell division by contacting cells with the bFGF muteins describedherein. Typically, stimulating cell division means that cells enter intoand complete mitosis. Practically, this means that the cell populationnumber doubles and increases exponentially. Contacting cells with themutein is accomplished by any means known to those skilled in the art.In one embodiment, contact may occur in vitro. This would beparticularly useful if an isolated population of a patient's or donor's(human or nonhuman) cells required expansion of cell number. In thisinstance the cells may be cultivated in vitro, by means well known tothose practicing the art, in a nutrient medium containing a sufficientquantity of one or more of the bFGF muteins. Preferably, the muteinwould be present at 0.001 picograms to 1000 micrograms per milliliter,more preferably at 0.001 nanograms to 1000 nanograms per milliliter andmost preferably at 0.01 nanograms to 100 nanograms per milliliterconcentration.

In another embodiment, contact may occur in vivo. In this preferredtherapeutic approach, an animal suffering from a wound, ischemia, heartdisease (e.g., coronary artery disease), peripheral vascular disease, agastric or duodenal ulcer, stroke or neural injury is treated byadministering to the animal a composition comprising one or more of theisolated bFGF muteins of the invention. The composition may be apharmaceutical composition comprising a therapeutically effective amountof one or more isolated bFGF muteins of the invention and optionally apharmaceutically acceptable carrier or excipient therefor. Theadministration of a bFGF composition promotes healing in conditions suchas a wound, ischemia, heart disease (e.g., coronary artery disease),peripheral vascular disease, a gastric or duodenal ulcer, stroke orneural injury through the mitogenic, trophic, angiogenic,neuroprotective or other inherent property of the bFGF muteins describedherein.

The bFGF mutein-containing compositions should be formulated and dosedin a fashion consistent with good medical practice, taking into accountthe clinical condition of the patient, the site of delivery of the bFGFmutein composition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The“therapeutically effective amount” of the bFGF mutein for purposesherein is thus determined by such considerations.

The bFGF muteins and the pharmaceutical compositions of the presentinvention may be administered by any means that achieve the generallyintended purpose: to promote the healing of tissue damaged throughtrauma or disease. For example, administration may be by oral, ocular,otical, rectal, parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, intravaginal, topical (as by powders, ointments, dropsor transdermal patch), bucal, intrathecal or intracranial routes, as anoral or nasal spray or as ocular or intraotic drops. The term“parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. Compositions within the scope ofthe invention include all compositions wherein a bFGF mutein is presentin an amount that is effective to achieve the desired medical effect fortreament of a wound, ischemia, heart disease (e.g., coronary arterydisease), peripheral vascular disease, a gastric or duodenal ulcer,stroke or neural injury. While individual needs may vary from onepatient to another, the determination of the optimal ranges of effectiveamounts of all of the component is within the ability of the clinicianof ordinary skill.

Generally, the formulations are prepared by contacting a bFGF mutein ofthe invention uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as areliposomes. Aqueous and non-aqueous vehicles would preferably bemaintained at a pH of about 3 to 10. Also, it will be understood thatthe use of certain of the foregoing excipients, carriers, or stabilizersmay result in the formation of bFGF mutein polypeptide salts.

For parenteral administration, in one embodiment, the bFGF muteins areformulated generally by mixing one or more of them at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

As a general proposition, the total therapeutically effective amount ofbFGF mutein administered parenterally per dose will be from about 0.001pg/kg body weight to about 10 mg/kg body weight daily and in most casesit would be administered in a dosage from about 1 pg/kg body weight to 5mg/kg body weight per day and preferably the dosage is from about 10pg/kg body weight to about 1 mg/kg body weight daily, taking intoaccount the routes of administration, symptoms, etc. If givencontinuously, the bFGF mutein(s) may be administered either by 1-10injections per day or by continuous subcutaneous infusion, for example,using a mini-pump. An intravenous bag solution may also be employed. Thekey factor in selecting an appropriate dose is the result obtained, asmeasured, for example, by increases in the circulating bFGF mutein levelor by determining the extent to which healing is promoted. Other usefulmeasures of determining therapeutic effectiveness are known to one ofordinary skill in the art. The length of treatment needed to observechanges, and the interval following treatment for responses to occur,may vary depending on the desired effect.

Pharmaceutical compositions for use in such methods comprise one or moreof the isolated bFGF muteins of the present invention and may optionallycomprise a pharmaceutically acceptable carrier or excipient therefor, asdescribed above.

In another embodiment of the invention, a localized, topical applicationof one or more of the bFGF muteins would be a particularly preferredroute of administration to promote the healing of a wound. Additionally,a localized application of one or more of the bFGF muteins may also beused in a treatment designed to promote the healing of damaged internaltissue or the growth of new tissue. For the purposes of thisapplication, internal tissue may refer to bone, tendon, cartilage,muscle, neural or other tissues and cells known by one skilled in theart to be responsive to basic Fibroblast Growth Factor. Damage mayresult from an extrinsic process such as an accident or surgery, oralternatively, damage may be the result of an intrinsic process, such asa disease, as what might be found in the case of heart disease (e.g.,coronary artery disease; myocardial infarction), peripheral vasculardisease, gastric and duodenal ulcers, stroke or neural disease processesas observed with Alzheimer's or Parkinson's Disease.

If a localized, external application is desired, then the bFGF muteinsmay be applied in pharmaceutical composition such as an ointment, gel,or other appropriately known carrier in according to guidelinesestablished with the previously described pharmaceutical carriers. Aninternally localized application of a bFGF mutein composition may beobtained by direct application of a previously described pharmaceuticalcomposition, or the bFGF mutein compositions may be administered by asustained-release system. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919; EP 0 058 481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U.,et al., Biopolymers 22:547-556 (1983), poly (2-hydroxyethylmethacrylate) (Langer, R., et al., J. Biomed. Mat. Res. 15:167-277(1981); Langer, R., Chem. Tech. 12:98-105 (1982), ethylene vinyl acetate(Langer et al, Id.) and poly-D(−)-3-hydroxybutyric acid (EP 0 133 988).Sustained-release bFGF mutein compositions may also include liposomallyentrapped or absorbed bFGF muteins, which may be prepared by any of avariety of methods that have been well-described in the art (See U.S.Pat. Nos. 4,485,045 and 4,544,545; Epstein et al., Proc. Natl. Acad.Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 0 036 676; EP 0 052 322; EP 0 088 046; EP0 102 324; EP 0 142 641; EP 0 143 949; DE 3,218,121; and JP 83-118008).

As a general rule, the therapeutic objective in a localized treatmentprotocol is to obtain a tissue concentration of the bFGF mutein that isten times the normal bFGF tissue concentration (5 to 50 ng per gm wetweight tissue) and one hundred times the normal serum level of bFGF(Buckley, R., Proc. Natl. Acad. Sci. (USA) 82:7340-7344 (1985)). Thus,the aforementioned slow release polymers can be designed by thoseskilled in the art with this target concentration in mind. In thetreatment of a wound, this would generally be accomplished through theapplication of a solution, ointment, gel, etc. containing 0.1 to 10percent bFGF mutein. Effectively, this means the application of 0.1 to100 micrograms of bFGF mutein(s) per square centimeter of wound area. Itis anticipated that the exact dosage will vary depending on thecircumstances; for example, the initial treatment for a third degree bumwould be about 100 μg/cm² and, as the condition improved, theapplication of bFGF mutein would be decreased to about 0.1 μg/cm².

In another embodiment of the invention, the bFGF muteins may be combinedin a pharmaceutical composition with another growth factor such asvascular endothelial cell growth factor, epidermal growth factor,platelet-derived growth factor, or any other cytokine known to oneskilled in the art which would promote the healing of the aforementionedtarget tissues.

The bFGF muteins to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes) or by gamma orultraviolet irradiation according to art-known techniques. TherapeuticbFGF mutein(s) polypeptide compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The therapeutic compositions comprising the bFGF muteins of theinvention ordinarily may be stored in unit or multi-dose containers, forexample, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous bFGF mutein solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized bFGF mutein using U.S.P. water that issuitable for injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainers can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, a muteinof the present invention may be employed in conjunction with othertherapeutic compounds.

In another embodiment of the invention, an animal suffering from awound, ischemia, heart disease (e.g., coronary artery disease),peripheral vascular disease, gastric or duodenal ulcers, stroke orneural injury may be treated by introducing into the animal one or moreof the isolated nucleic acid molecules of the invention comprising apolynucleotide encoding one or more of the bFGF muteins or a fragmentsthereof. This approach, known generically as “gene therapy”, generallytargets somatic cells as a means oftreating inherited diseases, cancerand acquired disorders (Bank, A., Bioessays 18(12):999-1007 (1996)).This embodiment is designed to increase the level of bFGF mutein geneexpression in and around the tissue and cells wherein the medicalobjective is to promote healing or new tissue growth.

Initial research in the area of gene therapy focused on a fewwell-characterized and highly publicized disorders: cysticfibrosis-Drumm, M. L. et al., Cell 62:1227-1233 (1990); Gregory, R. J.et al., Nature 347:358-363 (1990); Rich, D. P. et al., Nature347:358-363 (1990); and Gaucher disease-Sorge, J. et al., Proc. Natl.Acad. Sci. (USA) 84:906-909 (1987); Fink, J. K. et al., Proc. Natl.Acad. Sci. (USA) 87:2334-2338 (1990); and certain forms ofhemophilia-Bontempo, F. A. et al., Blood 69:1721-1724 (1987); Palmer, T.D. et al., Blood 73:438-445 (1989); Axelrod, J. H. et al., Proc. Natl.Acad. Sci. (USA) 87:5173-5177 (1990); Armentano, D. et al., Proc. Natl.Acad. Sci. (USA) 87:6141-6145 (1990); and muscular dystrophy-Partridge,T. A. et al., Nature 337:176-179 (1989); Law, P. K. et al., Lancet336:114-115 (1990); Morgan, J. E. et al., J. Cell Biol. 111:2437-2449(1990).

More recently, the application of gene therapy in the treatment of awider variety of disorders is progressing, for example: cancer(Runnebaum, I. B., Anticancer Res. 17(4B):2887-2890 (1997), heartdisease (Rader, D. J., Int. J. Clin. Lab. Res. 27(1):35-43 (1997);Malosky, S., Curr. Opin. Cardiol. 11(4):361-368 (1996)), central nervoussystem disorders and injuries (Yang, K., et al., Neurotrauma J.14(5):281-297 (1997); Zlokovic, B. V., et al., Neurosurgery40(4):789-803 (1997); Zlokovic, B. V., et al., Neurosurgery40(4):805-812 (1997), vascular diseases (Clowes, A. W., Thromb. Haemost.78(1):605-610 (1997), muscle disorders (Douglas, J. T., et al.,Neuromuscul. Disord. 7(5):284-298 (1997); Huard, J., et al.,Neuromuscul. Disord. 7(5):299-313 (1997)), rheumatoid arthritis (Evans,C. H., et al., Curr. Opin. Rheumatol. 8(3): 230-234 (1996)) andepithelial tissue disorders (Greenhalgh, D. A., et al., Invest Dermatol.J. 103(5 Suppl.):63S-93S (1994)).

In a preferred approach, one or more isolated nucleic acid molecules ofthe invention is introduced into or administered to the animal. Suchisolated nucleic acid molecules may be incorporated into a vector orvirion suitable for introducing the nucleic acid molecules into thecells or tissues of the animal to be treated, to form a transfectionvector. Techniques for the formation of vectors or virions comprisingthe bFGF mutein-encoding nucleic acid molecules are well-known in theart and are generally described in “Working Toward Human Gene Therapy,”Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J. D. et al., eds., NewYork: Scientific American Books, pp. 567-581 (1992). An overview ofsuitable vectors or virions is provided in an article by Wilson, J. M.,Clin. Exp. Immunol. 107(Suppl. 1):31-32 (1997)). Such vectors arederived from viruses that contain RNA (Vile, R. G., et al., Br. MedBull. 51(1):12-30 (1995)) or DNA (Ali M., et al., Gene Ther.1(6):367-384 (1994)). Example vector systems utilized in the art includethe following: retroviruses (Vile, R. G., supra.), adenoviruses (Brody,S. L. et al., Ann. N.Y. Acad. Sci. 716:90-101 (1994)),adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J.11(8):624-634 (1997), adeno-associated viruses (Flotte, T. R. andCarter, B. J., Gene Ther. 2(6):357-362 (1995)), herpes simplex virus(Latchman, D. S., Mol. Biotechnol. 2(2):179-195 (1994)), Parvovirus(Shaughnessy, E., et al., Semin Oncol. 23(1):159-171 (1996)) andreticuloendotheliosis virus (Donburg, R., Gene Therap. 2(5):301-310(1995)). Also of interest in the art, the development ofextrachromosomal replicating vectors for gene therapy (Calos, M. P.,Trends Genet. 12(11):463-466 (1996)).

Other, nonviral, methods for gene transfer known in the art (Abdallah,B., et al., Biol. Cell 85(1):1-7 (1995)) might be utilized for theintroduction of bFGF mutein polynucleotides into target cells; forexample, receptor-mediated DNA delivery (Philips, S. C., Biologicals23(1): 13-16 (1995)) and lipidic vector systems (Lee, R. J. and Huang,L., Crit. Rev. Ther. Drug Carrier Syst. 14(2):173-206 (1997)) arepromising alternatives to viral-based delivery systems.

General methods for construction of gene therapy vectors and theintroduction thereof into affected animals for therapeutic purposes maybe obtained in the above-referenced publications, the disclosures ofwhich are specifically incorporated herein by reference in theirentirety. In one such general method, vectors comprising the isolatedpolynucleotides of the present invention are directly introduced intotarget cells or tissues of the affected animal, preferably by injection,inhalation, ingestion or introduction into a mucous membrane viasolution; such an approach is generally referred to as “in vivo” genetherapy. Alternatively, cells, tissues or organs, particularly thoseassociated with a wound, ischemia, heart disease (e.g., coronary arterydisease), peripheral vascular disease, a gastric or duodenal ulcer,stroke or neural injury, may be removed from the affected animal andplaced into culture according to methods that are well-known to one ofordinary skill in the art; the vectors comprising the bFGF muteinpolynucleotides may then be introduced into these cells or tissues byany of the methods described generally above for introducing isolatedpolynucleotides into a cell or tissue, and, after a sufficient amount oftime to allow incorporation of the bFGF mutein polynucleotides, thecells or tissues may then be re-inserted into the affected animal. Sincethe introduction of the bFGF mutein gene is performed outside of thebody of the affected animal, this approach is generally referred to as“ex vivo” gene therapy.

For both in vivo and ex vivo gene therapy, the isolated bFGF muteinpolynucleotides of the invention may alternatively be operatively linkedto a regulatory DNA sequence, which may be a bFGF mutein promoter or anenhancer, or a heterologous regulatory DNA sequence such as a promoteror enhancer derived from a different gene, cell or organism, to form agenetic construct as described above. This genetic construct may then beinserted into a vector, which is then used in a gene therapy protocol.The need for transcriptionally targeted and regulatable vectorsproviding cell-type specific and inducible promoters is well recognizedin the art (Miller, N. and Whelan, J., Hum. Gene Therap. 8(7):803-815(1997); and Walther, W. and Stein, U., Mol. Med. J. 74(7):379-392(1996)), and for the purposes of bFGF mutein gene therapy, isincorporated herein by reference.

The construct/vector may be introduced into the animal by an in vivogene therapy approach, e.g., by direct injection into the target tissue,or into the cells or tissues of the affected animal in an ex vivoapproach. In another preferred embodiment, the genetic construct of theinvention may be introduced into the cells or tissues of the animal,either in vivo or ex vivo, in a molecular conjugate with a virus (e.g.,an adenovirus or an adeno-associated virus) or viral components (e.g.,viral capsid proteins; see WO 93/07283). Alternatively, transfected hostcells, which may be homologous or heterologous, may be encapsulatedwithin a semi-permeable barrier device and implanted into the affectedanimal, allowing passage of bFGF mutein polypeptides into the tissuesand circulation of the animal but preventing contact between theanimal's immune system and the transfected cells (see WO 93/09222).These approaches result in increased production of bFGF mutein by thetreated animal via (a) random insertion of the bFGF mutein gene into thehost cell genome; or (b) incorporation of the bFGF mutein gene into thenucleus of the cells where it may exist as an extrachromosomal geneticelement. General descriptions of such methods and approaches to genetherapy maybe found, forexample, in U.S. Pat. No. 5,578,461; WO94/12650; and WO 93/09222.

The methods of the invention are particularly well-suited for themedical treatment of a wound, ischemia, heart disease (e.g., coronaryartery disease), peripheral vascular disease, gastric or duodenalulcers, stroke or neural injury in any animal, preferably in mammals andmost particularly in humans. It will be readily apparent to one ofordinary skill in the relevant arts that other suitable modificationsand adaptations to the methods and applications described herein areobvious and may be made without departing from the scope of theinvention or any embodiment thereof. Having now described the presentinvention in detail, the same will be more clearly understood byreference to the following examples, which are included herewith forpurposes of illustration only and are not intended to be limiting of theinvention.

All patents and publications referred to herein are expresslyincorporated by reference.

EXAMPLES General Materials and Methods

Chemicals and Reagents: All restriction enzymes, T4 DNA ligase, T4polynucleotide kinase and Litmus vectors were purchased from New EnglandBiolabs (Beverly, Mass.). Oligonucleotide synthesis was provided byGibco BRL (Gaithersburg, Md.); pET expression vectors and BL21(DE3)competent cells were purchased from Novagen (Madison, Wis.), and theSculptor™ in vitro mutagenesis system was purchased from Amersham(Arlington Heights, Ill.). DNA preparations were accomplished usingQiagen products and protocols (Chatsworth, Calif.). Protease inhibitorsPMSF, leupeptin and pepstatin A were purchased from Sigma (St. Louis,Mo.), and DTT was purchased from Pierce (Rockford, Ill.). TheCellTiter®96 Aq_(ueous) Non-Radioactive Cell Proliferation Assay waspurchased from Promega (Madison, Wis.). Cell culture reagents werepurchased from Gibco (Grand Island, N.Y.).

Abbreviations: bFGF, basic fibroblast growth factor; DTT,Dithiothreitol; PMSF, phenylmethanesulfonyl fluoride; Hepes,-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; EDTA,ethylenediamine tetra acetic acid; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; IPTG, isopropylp-D-thiogalactopyranoside; DMEM, Dulbecco's Modified Eagle's Media

Example 1

Construction of bFGF Muteins

A synthetic gene for human basic fibroblast growth factor (bFGF) waspurchased from R&D Systems (Minneapolis, Minn.) and is based on thepublished cDNA sequence (J. A. Abraham, EMBO J. 5(10):2523-2328 (1986)).Numerous modifications of this gene were made by site-directedmutagenesis to incorporate unique restriction enzyme sites. In all casesthe translated amino acid sequence was unaltered. The resultant DNA andamino acid sequence is found in FIG. 1.

Site-Directed Mutagenesis of the bFGF gene was accomplished bysubcloning the bFGF sequence into the phagemid vector Litmus 28 (NewEngland Biolabs, Beverly, Mass.). Single stranded DNA template waspurified from phage following infection with helper phage. The initialmutations at amino acid positions E89 and D101 were made by singlestranded DNA site-directed mutagenesis using the Sculptor™ in vitromutagenesis system (Amersham, Arlington Heights, Ill.) according to themanufacturer's recommendations. The E89A, E89Y and D101A single sitemutations were achieved using this method. Mutant DNA constructs werethen subcloned back into the pET vector for expression of protein (seebelow).

All other mutations reported in Table 1 were constructed using cassettemutagenesis. The L137A mutation was made by inserting complementaryoligonucleotides into the AflII and ApaI sites in which the CTT codonfor leucine was replaced with the GCT codon for alanine (FIG. 1). AHindIII site (not shown) was removed for convenient screening ofpositives. The DNA sequence for the above mentioned mutations differssomewhat from that which appears in FIG. 1 although the amino acidsequence remains identical except for the introduced site-directedmutations. The DNA sequence for the E89A, E89Y, D101A and L137A mutantsdiffers from that reported in FIG. 1 only between the KpnI and ApaIrestriction enzyme sites. Except for the introduced mutation (boxedcodon in FIG. 1) the following DNA sequence between the KpnI and ApaIsites is representative for the E89A, E89Y, D101A and L137A mutants:CTGGCTATGAAGGAAGATGGAAGATTACTGGCTTCTAAATGTGTTACGGATGAGTGTTTCTTTTTTGAACGATTGGAATCTAATAACTACAATACTTACCGCTCGAGAAAATACACCAGTTGGTATGTGGCACTTAAGCGTACCGGTCAGTACAAGCTTGGTTCTAAAACGGGCC (SEQ ID NO:3). The E89A and D101Adouble mutation reported in Table I was constructed by insertingcomplementary oligonucleotides into the KpnI and XhoI sites (FIG. 1).The E89A, D101A, L137A triple mutant was constructed by combining theE89A, D101A double mutant with the single L 137A mutant using therestriction enzyme sites engineered into the DNA sequence (FIG. 1). Allmutations were confirmed by DNA sequencing of plasmid DNA. Cassettemutagenesis was performed using the bFGF expression construct (seebelow) such that no further subdloning was required.

Example 2

Expression and Purification of bFGF Muteins

Expression of wild-type and mutant proteins was performed using amodified pET expression vector as reported by Squires et al., J. Biol.Chem. 263: 16297-16302 (1989)) using the two-cistron method originallyreported by Schoner et al., Proc. Natl. Acad. Sci. (USA) 83:8506-8510(1986)). DNA was transformed into competent BL21(D.E3) Escherichia coli,and single colonies were chosen for larger scale growth. Fermentationswere either done on a 5 Liter scale with a BioFlo II fermenter from NewBrunswick at the Bioprocessing Resource Center at Penn State University(University Park, Pa.) or in house using 2 Liter baffled shake flasks.

When fermented in the New Brunswick fermenter, 50 ml Luria Broth (10 gmtryptone, 5 gm yeast extract, 5 gm NaCl per liter) containing 50 ug/mlcarbenicillin was inoculated with the construct of choice transformedinto BL21(DE3) and grown overnight. The 50 ml overnight culture was thenused to inoculate a 5 liter volume of Super Broth (35 gm tryptone, 20 gmyeast extract, 5 gm NaCI per liter) containing 50 ug/ml ofcarbenicillin. Growth was continued until the OD_(600mm) reached between8-10 at which time IPTG (isopropyl β-D-thiogalactopyranoside) was addedto a final concentration of 1.0 mM to induce expression of the T7polymerase gene from BL21 (DE3) cells. Cells were harvested 5 hours postinduction using a continuous flow centrifuge (T-1-P Sharples) and theresulting paste was stored at −70° C.

When grown in 2 L baffled shake flasks, a 250 ml culture containing theconstruct of interest was grown overnight in a 500 ml baffled shakeflask in Terrific Broth (12 gm tryptone, 24 gm yeast extract, 4 mlglycerol per liter) containing 50 ug/ml carbenicillin. The next day 2 Lbaffled shake flasks containing 800 ml of Terrific Broth with 100 ug/mlampicillin and 0.2% glucose were inoculated with 12 ml of the overnightculture and grown in a New Brunswick incubator shaker at 240 rpm untilthe OD_(600mm) reached 0.8 at which time IPTG was added to a finalconcentration of 10 mM. Cultures were allowed to grow an additional 5hours post induction. Cells were then harvested by centrifugation andstored at −70° C. At all times cultures were grown at 30° C. to permitexpression of bFGF in the soluble E. coli fraction.

Mutant and wild-type proteins were purified by first resuspending E.coli cell paste (stored at −70° C.) in load buffer (50 mM HEPES, pH 7.5,100 mM NaCl, 10 mM Dithiothreitol (DTT), 1 mM EDTA) to which thefollowing protease inhibitors were added: 1 mM PMSF, 1 ug/ml pepstatin Aand 1 ug/ml leupeptin. Typically, 50 gm of cell paste was resuspended in300 ml buffer and sonicated on ice using a Branson Ultrasonics Sonifier450 (Danbury, Conn.) until cells were completely lysed. The lysed cellsuspension was then centrifuged at 4° C. in a Beckman Avanti™ J-25centrifuge at 75,000×g for 30 min. Supernatants were combined andfiltered through a 0.8 uM filter. The entire sample was then applied at3 ml/min to a 5 ml HiTrap SP column (Pharmacia Biotech, Uppsala, Sweden)previously equilibrated with load buffer and attached to a GradiFracSystem (Pharmacia Biotech, Uppsala, Sweden). Following sample load thecolumn was washed with load buffer until the A₂₈₀ of the eluate returnedto baseline. A 5 ml HiTrap Heparin was attached in tandem to the SPcolumn and bound protein was bumped from the SP column to the Heparincolumn using 0.6 M NaCl in load buffer. Under these conditions bFGF iseluted from the SP resin and binds specifically to the Heparin affinitycolumn. Following an extensive wash of the HiTrap Heparin column using0.6 M NaCl to remove non-specifically bound protein, bFGF is eluted witha 0.6 M NaCl to 3.0 M NaCl linear gradient. Wild-type and mutantproteins were routinely purified to >95% using this procedure asdetermined by SDS-PAGE. In addition, all mutant proteins eluted at thesame NaCl concentration as wild-type bFGF indicating that the mutationshad not altered the affinity for heparin. We have previously reportedthat there is a good correlation between NaCl elution from a heparinaffinity column and Kd for heparin binding to bFGF measured byisothermal titration calorimetry (Thompson et al., Biochemistry33:3831-3840(1994)). A similarbinding affinity for heparin for each ofthese mutants compared to the wild-type protein is also independentconfirmation that no gross structural perturbations resulted due to theintroduced mutation(s).

Protein concentrations were determined by measuring the absorbance at280 nm and using an extinction coefficient of 16,766 M⁻¹ cm⁻¹ asreported by Pantoliano et al., Biochemistry 33:10229-10248 (1994)). Formutations in which an additional tyrosine was introduced uponsite-directed mutation the extinction coefficient was calculated byaddition of the individual extinction coefficients for tryptophans andtyrosines.

Example 3

bFGF mutein Mitogenicity

In order to demonstrate superagonist, mitogenic activity of the bFGFmutant proteins, experiments were undertaken to directly compare muteinand wild-type bFGF stimulation of fibroblast growth. NIH 3T3 mouseembryo fibroblast (1658-CRL), 3T3 Swiss albino mouse fibroblast (CCL-92)and Balb/c 3T3 clone A31 mouse embryo fibroblast (9163-CCL) cell lineswere obtained from the ATCC (American Type Culture Collection,Rockville, Md.). Cells were maintained in T-75 or T-150 tissue cultureflasks in Dulbecco's modified Eagle's media (DMEM) containingL-Glutamine, low glucose, 110 mg/l sodium pyruvate, pyridoxinehydrochloride and supplemented with 10% (v/v) heat-inactivated calfserum (Gibco, Grand Island, N.Y.). For assays, the cells were seeded in96 well plates in Dulbecco's modified Eagle's medium nutrient mixtureF-12 Ham (DMEM/F 12 1:1 mixture) containing L-glutamine, 15 mM Hepes,sodium bicarbonate, and lacking phenol red (Sigma, St. Louis, Mo.)supplemented with 0.5% or 1.0% (v/v) calf serum.

Promotion of cell proliferation for the various fibroblast cell lineswas determined using the CellTiter 96® AQUeous Non-Radioactive CellProliferation Assay (Promega, Madison, Wis.). This assay reliesonmitochondrial dehydrogenase enzymes to reduce a soluble tetrazoliumcompound into formazan in the presence of an electron coupling reagent(Dunigan et. al., Biotechniques 19:640-649 (1995)). The quantity offormazan product is directly proportional to the number of viable cellsin culture and is easily monitored at an absorbance of 490 nm. Thismethod has been shown to be equivalent to other methods of quantitatingcell proliferation (Gieni et al., Immun. Methods J. 187:85-93 (1995);Roehm et al., Immun. Methods J. 142: 257-265 (1991); Drummond et al.,Biochem. Biophys. Methods J. 31:123-134 (1996)).

Assays were performed in 96 well plates according to the manufacturer'sprotocol (Promega, Technical Bulletin #169) using either Falcon #3072 orCostar #3596 low evaporation lid tissue culture plates (FisherScientific, Pittsburgh, Pa.). Dilutions (typically ranging from 100 to0.001 ng/ml) of wild-type and site-directed mutant bFGFs to be assayedwere made serially in 96 well plates using low serum media and thenwarmed to, 37° C. Fibroblast cells were detached from T-150 plates byuse of 0.25% trypsin (Gibco, Grand Island, N.Y.) for 3 minutes. Trypsinwas inactivated by addition of DMEM supplemented with 10% calf serum andresting for 1 hr. Cells were then washed twice with low serum media,counted, and plated at a density of 5×10³ cells/well into the 96-wellplates containing dilutions of bFGF. Cells were incubated in thepresence of bFGFs for 18-20 h at 37° C. under 5% CO₂ and plates wereprocessed according to the manufacturer's recommendations. Absorbancewas measured at 490 nm (minus 700 nm to correct for background) using aSPECTRAmax™ 250 Microplate Spectrophotometer (Molecular Devices,Sunnyvale Calif.). Each dilution was run in quadruplicate and all plateshad a negative control also in quadruplicate (cells in low serum mediumwith no added growth factor) which was subtracted from the mean of eachquadruplicate value.

An exemplary experiment is presented in FIG. 2, demonstrating the lowerED₅₀ values of the bFGF muteins. Data from experiments similar to whatis presented in FIG. 2 were utilized to construct Table 1. Mutantdesignations are as described in the detailed description of FIG. 2, andED₅₀ is defined as the concentration of growth factor required toachieve ½ maximal stimulation of a defined cell line.

Below in Table 1, superagonist mitogenicity of the bFGF muteins isdemonstrated. Mutein ED₅₀ values are reported as ratios to wild-typebFGF to compensate for assay to assay variation and to provide a measureof bFGF mutein mitogenicity relative to wild-type bFGF. Values reportedwere obtained utilizing recombinant wild-type bFGF expressed andpurified from E. coli and mutant forms of bFGF expressed and purified inan identical manner to the wild-type protein. Values reflect X-foldenhanced activity over the wild-type protein (X=the ratio reported inthe Table 1). n refers to the number of experiments performed. For allmutant bFGF data reported in Table 1, three fibroblast cell lines wereused (Swiss 3T3, NIH 3T3 and Balb/c 3T3). Although the absolute ED₅₀values varied for wild-type bFGF and muteins, the ratio of wild-typeED₅₀/mutant ED₅₀ remained consistent for the three cell lines tested.

TABLE 1 Mitogenic Activity of bFGF Superagonists Wild-type bFGF ED₅₀/ n(number of Mutant bFGF Mutant bFGF ED₅₀ experiments) E89A 2.8 42 E89Y13.0 40 D101A 4.4 26 E89A, D101A 14.3 10 E89A, D101A, L137A 7.3 12 L137A2.5 25

As is evidenced in the data presented in FIG. 2 and Table 1, all of themutant bFGF proteins are significantly more mitogenic than the wild-typebFGF protein.

3 1 483 DNA Homo sapiens CDS (1)..(474) 1 atg ggc acc atg gca gcc gggagc atc acc acg ctg ccc gcc ctt ccg 48 Met Gly Thr Met Ala Ala Gly SerIle Thr Thr Leu Pro Ala Leu Pro 1 5 10 15 gag gat ggc ggc agc ggc gccttc ccg ccc ggg cac ttc aag gac ccc 96 Glu Asp Gly Gly Ser Gly Ala PhePro Pro Gly His Phe Lys Asp Pro 20 25 30 aag cgg ctg tac tgc aaa aac gggggc ttc ttc ctg cgc atc cac ccc 144 Lys Arg Leu Tyr Cys Lys Asn Gly GlyPhe Phe Leu Arg Ile His Pro 35 40 45 gac ggc cga gtt gac ggg gtc cgg gagaag agc gac cct cac atc aag 192 Asp Gly Arg Val Asp Gly Val Arg Glu LysSer Asp Pro His Ile Lys 50 55 60 cta caa ctt caa gca gaa gag aga gga gttgtg tct atc aaa gga gtg 240 Leu Gln Leu Gln Ala Glu Glu Arg Gly Val ValSer Ile Lys Gly Val 65 70 75 80 tgt gct aac cgg tac ctg gct atg aaa gaagat ggt cga ctg ctg gct 288 Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu AspGly Arg Leu Leu Ala 85 90 95 tct aaa tgc gtt acc gat gag tgc ttc ttc ttcgaa cgt ctc gag tct 336 Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe GluArg Leu Glu Ser 100 105 110 aac aac tac aac acc tac cgt tcg aga aaa tacacc agt tgg tat gtg 384 Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr ThrSer Trp Tyr Val 115 120 125 gca ctt aag cgt acc ggt cag tac aaa ctt ggttct aag acg ggc cca 432 Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly SerLys Thr Gly Pro 130 135 140 ggt cag aaa gct atc ctg ttc ctg ccg atg tctgct aaa tct tgaggatcc 483 Gly Gln Lys Ala Ile Leu Phe Leu Pro Met SerAla Lys Ser 145 150 155 2 158 PRT Homo sapiens 2 Met Gly Thr Met Ala AlaGly Ser Ile Thr Thr Leu Pro Ala Leu Pro 1 5 10 15 Glu Asp Gly Gly SerGly Ala Phe Pro Pro Gly His Phe Lys Asp Pro 20 25 30 Lys Arg Leu Tyr CysLys Asn Gly Gly Phe Phe Leu Arg Ile His Pro 35 40 45 Asp Gly Arg Val AspGly Val Arg Glu Lys Ser Asp Pro His Ile Lys 50 55 60 Leu Gln Leu Gln AlaGlu Glu Arg Gly Val Val Ser Ile Lys Gly Val 65 70 75 80 Cys Ala Asn ArgTyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala 85 90 95 Ser Lys Cys ValThr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser 100 105 110 Asn Asn TyrAsn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val 115 120 125 Ala LeuLys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro 130 135 140 GlyGln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 3 175DNA Homo sapiens 3 ctggctatga aggaagatgg aagattactg gcttctaaatgtgttacgga tgagtgtttc 60 ttttttgaac gattggaatc taataactac aatacttaccgctcgagaaa atacaccagt 120 tggtatgtgg cacttaagcg taccggtcag tacaagcttggttctaaaac gggcc 175

What is claimed is:
 1. A mutein of human basic fibroblast growth factor,or a biologically active peptide thereof, comprising one or more of thefollowing substitutions: (a) substitution of Glutamate 89 with alanineor tyrosine; (b) substitution of Aspartate 101 with alanine; or (c)substitution of Leucine 137 with alanine; or any combination thereof,wherein the numbering of amino acids is based on SEQ ID NO:1.
 2. Themutein of claim 1 which is human basic fibroblast growth factor [Ala⁸⁹].3. The mutein of claim 1 which is human basic fibroblast growth factor[Ala¹⁰¹].
 4. The mutein of claim 1 which is human basic fibroblastgrowth factor [Ala¹³⁷].
 5. The mutein of claim 1 which is human basicfibroblast growth factor [Ala^(89, 101)].
 6. The mutein of claim 1 whichis human basic fibroblast growth factor [Ala^(89, 137)].
 7. The muteinof claim 1 which is human basic fibroblast growth factor[Ala^(101, 137)].
 8. The mutein of claim 1 which is human basicfibroblast growth factor [Ala^(89, 101, 137)].
 9. The mutein of claim 1which is human basic fibroblast growth factor [Tyr⁸⁹].